1. Volume 97, Issue 2, Pages 233-244 (April 1999)
Exit from Mitosis Is Triggered by Tem1-Dependent Release of the Protein Phosphatase Cdc14 from Nucleolar RENT Complex 
Wenying Shou, Jae Hong Seol, Anna Shevchenko, Christopher Baskerville, Danesh Moazed, Z.W.Susan Chen, Joanne Jang, Andrej Shevchenko, Harry Charbonneau, Raymond J Deshaies 
Cell 
Volume 97, Issue 2, Pages (April 1999)
DOI: /S (00)

2. Figure 3
Loss of Silencing Fails to Bypass the Essential Requirement for TEM1
Wild-type (+), net1-1, sir2Δ, sir3Δ, or sir4Δ cells that carried tem1Δ and were sustained by a [GAL1-TEM1, URA3] plasmid were grown on YPG (TEM1 expressed) and then plated onto either YPG (right panel) or synthetic glucose medium supplemented with 5-FOA (left panel) to select for colonies that were able to grow in the absence of the [GAL1-TEM1, URA3] plasmid. The plates were incubated at 25°C and photographed after 1 (YPGal plate) or 2 weeks (5-FOA plate).
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3. Figure 1
net1-1 Bypasses tem1Δ by Inducing Ectopic Clb2 Degradation and Sic1 Accumulation
tem1Δ::GAL1-UPL-TEM1 net1-1 and tem1Δ::GAL1-UPL-TEM1 cells grown in YPG (TEM1 expressed) at 25°C were arrested in G1 phase with α factor and released into YPD (TEM1 repressed) at time = 0.
(A) At 0, 1, 2, 4, and 12 hr, samples were taken to measure budding index.
(B) Same as (A), except that at either 2 hr (NET1) or 3 hr (net1-1) following release from α factor arrest, α factor was added back to prevent cells from proceeding through a second cell cycle. At the indicated time points (hr), samples were taken to measure Clb2, Sic1, and Cdc28 protein levels by immunoblotting.
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4. Figure 2 Identification of the RENT Complex
(A) Purification of Net1. Extracts (35 mg) of myc9-NET1 (lane 2) and untagged control (lane 1) strains were fractionated on beads containing covalently linked anti-Myc monoclonal antibody 9E10. Eluted proteins were separated on a 10%–15% SDS-polyacrylamide gradient gel and visualized by silver staining. Protein bands specifically detected in the immunoprecipitates from myc9-NET1 but not the untagged control strain were excised and identified by mass spectrometry.
(B) Protein identification by high mass accuracy MALDI peptide mass mapping. Mass spectrum acquired from a 0.5 μL aliquot of in-gel digest of the 62 kDa band revealed 13 peptide ions that matched the calculated masses of protonated tryptic peptides from Cdc14 with accuracy better than 50 ppm (designated with asterisks). These peptides covered more than 26% of the protein sequence. Ions originating from the matrix are designated by “M.” Peptide ions of known trypsin autolysis products are also marked. The inset shows an isotopically resolved peptide ion having the monoisotopic weight Mass resolution was better than 7000 FWHM (full width at half maximum) despite the very low amount of protein present in the gel.
(C) Net1 associates with Cdc14. Extracts from strains with the indicated genotypes were immunoprecipitated with either 9E10 antibodies or anti-haemagglutinin (HA) monoclonal antibodies 12CA5. The immunoprecipitates (IP) and the input extracts were immunoblotted with 9E10 (top panel) and 12CA5 (bottom panel) to detect Net1 and Cdc14 proteins, respectively.
(D) Net1-dependent association between Sir2 and Cdc14. Extracts of CDC14-HA3 cells with the indicated genotypes were immunoprecipitated with anti-Sir2 antibodies. Δ, strains deleted for SIR2 or NET1; +, wild type; −1, the net1-1 allele. The immunoprecipitates (IP) and the input extracts were immunoblotted with 12CA5 (top panel) and anti-Sir2 antibodies (bottom panel) to detect the Cdc14 and Sir2 proteins, respectively.
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5. Figure 4 Net1 Is an Inhibitor of and a Candidate Substrate for Cdc14
(A and B) Cdc14 activity is elevated in net1 mutants. Cdc14-HA3 was retrieved from lysates of the indicated strains (in the “14-HA3” column: −, untagged; +, CDC14-HA3; in the “NET1” column: +, NET1; −1, net1-1; Δ, net1Δ) by immunoprecipitation with 12CA5 antibodies and was incubated with 32P-Sic1 for 20 min at 22°C. The specific activity of Cdc14-HA3 in each reaction was determined by measuring trichloroacetic acid–soluble counts released during the incubation and dividing by the relative amount of Cdc14-HA3 as determined by immunoblotting. All values were corrected by subtracting out background signals obtained with untagged control sample; relative amounts of proteins were normalized such that the specific activity of Cdc14 was 1.0 in wild-type cells. The background signals were ∼350 and ∼27 in the 32P release and immunoblot assays, respectively. The results of duplicate reactions are presented. In (A), asynchronous cells were used, whereas in (B), cells were synchronized in G1 phase with α factor prior to analysis.
(C) Net1 inhibits Cdc14 phosphatase activity in vitro. Extracts of myc9-NET1 cells were immunoprecipitated with 9E10 antibodies, and protein A beads were coated with anti-GST antibodies to prepare Net1 and α-GST beads, respectively. Both matrices were washed extensively and incubated with wild-type or mutant GST-Cdc14 purified from E. coli (20 ng for GST beads and 60 ng for Net1 beads). The specific activity of bead-bound GST-Cdc14 was evaluated as described above using 32P-Sic1 as the substrate. Levels of GST-Cdc14 recruited to the Net1 and α-GST beads were assessed by immunoblotting with α-GST antibodies. The activity of mutant Cdc14 bound to the α-GST beads was used for background correction. For simplicity, in (B) and (C) only the final values (32P-Sic1 dephosphorylation divided by relative amount of Cdc14 antigen) are shown.
(D and E) Net1 is a candidate substrate for Cdc14. (D) Net1 is a phosphoprotein in vivo. myc9-NET1 (lane 1) and untagged (lane 2) strains were labeled with 32P-inorganic phosphate. Extracts of labeled cells were immunoprecipitated with 9E10 antibodies, and recovered proteins were fractionated by SDS-PAGE and detected by autoradiography. (E) Net1 can be dephosphorylated by Cdc14 in vitro. CDC14 myc9-NET1 (+, lanes 1 and 2) and cdc14-1 myc9-NET1 (cdc14-1, lanes 3–6) cells were grown at 25°C or shifted to 37°C for 3 hr, as indicated. Myc9-Net1 was immunoprecipitated from cell extracts with 9E10 antibodies, and portions of the Net1 immunoprecipitate from the 37°C cdc14-1 culture were either left untreated (lane 4) or incubated with wild-type (A; lane 5) or C283S mutant (I; lane 6) GST-Cdc14 for 60 min at 30°C. All immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with 9E10 to detect Myc9-Net1.
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6. Figure 5 Net1-Dependent Localization of Cdc14 to the Nucleolus
(A) Net1 and Cdc14 both localize to the nucleolus. Top row: untagged wild-type cells were probed with 9E10 antibodies or anti-hemagglutinin monoclonal antibody HA.11 plus the DNA-binding dye DAPI. Center and bottom rows: cells with the indicated genotype were probed with monoclonal antibodies (9E10 or HA.11, column 1) and rabbit anti-RPA 190 antibodies (column 2). The third column shows a merge of the two antibody staining patterns in columns 1 and 2. The fourth column shows the position of nuclei, as revealed by DAPI staining.
(B) Untagged control (top row) and CDC14-HA3 cells carrying NET1, net1-1, or net1Δ alleles (rows 2–4, respectively) were stained with HA.11 antibodies (column 1) and DAPI (column 2).
(C) Untagged control (top row) and net1Δ CDC14-HA3 (bottom row) cells were stained with HA.11 antibodies (column 1) and DAPI (column 2). DIC images are shown in column 3 to reveal the outline of cells.
(D) The levels of Cdc14-HA3 and Cdc28 proteins in the indicated strains were evaluated by immunoblotting with 12CA5 antibodies. The relative levels of Cdc14-HA3 ([14]) are indicated below the top panel.
Cell  , DOI: ( /S (00) )

7. Figure 6
Cdc14 Is Released from the Nucleolus during Anaphase/Telophase
(A) Asynchronous myc9-NET1 CDC14-HA3 cells were double labeled with rabbit anti-tubulin antibodies (column 2) and one of the following mouse antibodies (column 1): anti-Nop1 (top panel), 9E10 (center panel), or HA.11 (bottom panel). The positions of nuclei, as revealed by DAPI staining, are shown in Column 3.
(B) Cdc14-myc9/CDC14 diploid cells were probed with 9E10, DAPI, and anti-tubulin antibodies (columns 1–3, respectively). DIC images are shown in column 4. Cdc14 exhibits three different types of staining patterns: delocalized over the entire nucleus (center row), the entire cell (top row) during anaphase/telophase, or restricted to the nucleolus (bottom row) during interphase. Note the absence of extranucleolar Cdc14 in the G1 cell shown in the bottom row.
(C) myc9-NET1 CDC14-HA3 cells were synchronized with α factor and then released into YPD at time = 0. Samples withdrawn and fixed at the indicated time points were double labeled with anti-tubulin and either 9E10 (to detect Myc9-Net1) or HA.11 (to detect Cdc14-HA3) antibodies. For each sample, more than 200 cells were counted to calculate the percentage of cells with delocalized Cdc14 or Net1 and the percentage of cells with long spindles.
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8. Figure 7 Release of Cdc14 from the Nucleolus Requires Tem1
(A) tem1Δ::GAL1-UPL-TEM1 CDC14-HA3 cells grown in YPG (TEM1 expressed) were either sampled directly (top panel) or arrested in G1 phase with α factor and subsequently released into YPD (TEM1 repressed) for 3 hr (bottom panel). Cells were double labeled with HA.11 to visualize Cdc14-HA3 (column 1) and either anti-RPA190 or anti-tubulin antibodies to visualize nucleoli or mitotic spindles, respectively (column 2).
(B) A similar block/release protocol was conducted with a tem1Δ::GAL1-UPL-TEM1 CDC14-HA3 net1-1 strain, except that samples were processed for indirect immunofluorescence 4–6 hr after release from α factor arrest. A longer release time was required for net1-1 strains to progress from G1 to anaphase/telophase due to the ∼2-fold longer doubling time of this mutant (W. S., data not shown). The percentages of cells with long spindles and focal Cdc14-HA3 staining were quantitated for this sample as well as the samples from (A). More than 50 cells were counted for each sample.
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9. Figure 8 The “RENT Control” Hypothesis See text for details.
Cell  , DOI: ( /S (00) )